Cytokine Release Syndrome

Freddy Tadros and I are studying the efficacy of Chimeric Antigen Receptors (CARs) in the treatment of Acute Lymphoblastic Leukemia (ALL). To give just a brief overview of the treatment, scientists modify a person's own T-cells to express antibodies that target antigens present on lymphocytes that are involved with the leukemia. These lymphocytes are then destroyed by the body's own immune system (3). This treatment has had great success in clinical trials, with 5 of 5 participants experiencing a complete remission for 12 months, which was the period of time that the individuals had been monitored prior to publishing the paper, so these patients could be experiencing remission for even longer (2). Unfortunately, this promising treatment does have two main drawbacks: the cytokine release syndrome and B-cell aplasia. This blog will focus on the cytokine release syndrome.

Background
All of the patients that volunteered for treatment with CARs experienced the cytokine release syndrome (2). This entails the massive release of IL-6, TNF-alpha, and INF-gamma, all of which are different subclasses of the general category of cytokines. An interesting note here is that this generally only happens after the first dose of treatment; after that, the body is able to mediate the release. IL-6, in particular, is responsible for the inflammation that is associated with the first dose of this treatment (2). Symptoms of this can include fever, nausea, and scratchy throat; however, more severe cases result in dangerously high fevers and significant drops in blood pressure. Physicians have been able to control the cytokine release by use of steroids, which are generally sufficient to alleviate the symptoms. More drastic IL-6 increases requires more potent medication, the most popular of which is called Actemra, which actually blocks IL-6 activity and nulls the effects of it (2). Many scientists have been interested in the origin and mechanism of release of these cytokines so that their release can be more effectively controlled. The study I found evaluated the release of all three cytokines that are significant in the effect: IL-6, TNF-alpha, and INF-gamma.

The Culprit
The researchers studied the three different types of cells that could be responsible for the cytokine release, which are lymphocytes, monocytes, and granulocytes. They incubated a culture of each type of cell with an antibody specific to a cell surface receptor present on all three cell types previously listed. Of the three cells, the only one to release a significant amount of cytokines was the lymphocyte. They found that this one cell type was responsible for about 20% of the total TNF-alpha release that occurs in response to treatment. Granulocytes were found to secrete substances as well, but these were not relevant to the cytokine release syndrome (1). The graph at the right may deceive some to believe that both lymphocytes and monocytes are responsible for cytokine release due to both having a concentration of TNF-alpha of about 5000 pg/mL in the presence of PMA or LPS, which are the positive control substances, depending on cell type.It is important to note; however, that this is only due to the presence of a positive control substance which is inducing the cells to secrete the cytokine. The bar above the 1-H is the one that is significant in the discovery that only lymphocytes secrete TNF-alpha because the 1-H is the marker for the type of antibody that is used in treatment of individuals with ALL. The bottom graph confirms that lymphocytes are responsible for the release, rather than some secondary stimulation from monocytes (1). This is shown through lymphocytes alone having a secreted TNF-alpha concentration of around 750 pg/mL while the mixture of monocytes and lymphocytes had about 400 pg/mL.

Cytokine Release
These researchers found that the cytokine release syndrome is specific to the antibody type. This was established by the observation that TNF-alpha and INF-gamma were detected in their cultures of cells only 2 hours after administration of the CD52 antibody. They then repeated this experiment using an antibody that targeted CD4, which is another cell surface receptor on the cells that they were studying. They did not observe any significant release of cytokines when the cells were subjected to this antibody (1).

Another significant finding was that the cytokine release was not a result of activation of the innate immune system. This was discovered through 2 main experiments: one was the mutation of the tail of the antibody used to target the cells and the other was the mutation of an antibody found in the innate immune system. The first experiment, mutation of the tail of the antibody used for targeting the cells, resulted in a a 40-50% reduction in release of cytokines, indicating that it is a significant part of the cytokine release pathway. The second experiment, mutation of an antibody in the innate immune system, actually resulted in an increase in cytokines. This shows that it cannot be involved in the pathway because when the antibody is effectively nullified, the cytokine release syndrome is more significant (1).

This is the data that led the researchers to their conclusion that the cytokine release syndrome was not a result of innate immune system activation. The IgG1 C1q- is the mutated innate immune system antibody. In each of the 3 experiments, almost 2 times higher cytokine release was noted with this mutated antibody, indicating that the innate immune system cannot be responsible for the cytokine release. The researchers then mutated the antibody that is significant to the cytokine release syndrome because it is present on lymphocytes, denoted here as IgG1 Fc-. When this was mutated, the cytokine release decreased by almost 50% in the second 2 experiments relative to the IgG1 control. In the first experiment, the IgG1 control and mutated IgG1 Fc- exhibited relatively similar TNF-alpha release.

The researchers also found the receptor that is indicated in the cytokine release syndrome, and it is CD16, not CD52 as previously believed. Cytotoxic lymphocytes, or Natural Killer cells, demonstrated significant TNF-alpha release only when stimulated by CD16 antibody, not CD52. CD16 is expressed only on the surface of these natural killer cells, so this could not be a result from any other cells (1).

 The graph at the right demonstrates the above finding. In section B of the graph, Natural Killer cells subjected to 1-H, the antibody used throughout the study to elicit cytokine release, had very high concentrations of released TNF-alpha. The two variants, with and without CD16 antibody, showed drastic differences in TNF-alpha release. Without anti-CD16, NK cells secreted about 1000 pg/mL of TNF-alpha. With the anti-CD16, the cells only secreted about 250 pg/mL of TNF-alpha. This shows that when the receptor CD16 is nullified, the NK cells do not exhibit cytokine release, indicating that this receptor is significantly involved in the cytokine release syndrome.





Conclusion
All of the above experiments were designed to gradually limit the number of possible sources of the cytokine release syndrome. The researchers were able to narrow it down to the three cell types, then were able to isolate the specific surface protein involved in the release pathway. The only possibility for the source of the massive cytokine release is the Natural Killer cell stimulation by CD16 antibodies. This information will be able to aid other scientists in the creation of treatments that do not stimulate CD16 so as to reduce the effect of cytokine release. Another possible outcome of this research is a treatment that would focus on the cell surface receptor itself. Perhaps researchers could find a molecule that is able to block CD16 activation of Natural Killer cells. However; for the time being, we are limited to treating the symptoms of cytokine release rather than the source of them.

References
1. Wing, Mark G., Thibault Moreau, Judith Greenwood, Richard Smith, Geoff Hale, John Isaacs, Herman Waldmann, Peter Lachmann, and Alastair Compston. "Mechanism of First-Dose Cytokine-Release Syndrome by CAMPATH 1-H: Involvement of CD16 (Fc GRIII) and CD11a/CD18 (LFA-1) on NK Cells." Journal of Clinical Investigation (n.d.): n. pag. Web.

2.  Brentjens, R. J., M. L. Davila, I. Riviere, J. Park, X. Wang, L. G. Cowell, S. Bartido, J. Stefanski, C. Taylor, M. Olszewska, O. Borquez-Ojeda, J. Qu, T. Wasielewska, Q. He, Y. Bernal, I. V. Rijo, C. Hedvat, R. Kobos, K. Curran, P. Steinherz, J. Jurcic, T. Rosenblat, P. Maslak, M. Frattini, and M. Sadelain. "CD19-Targeted T Cells Rapidly Induce Molecular Remissions in Adults with Chemotherapy-Refractory Acute Lymphoblastic Leukemia." Science Translational Medicine 5.177 (2013): 177ra38. Print

3. "Cancer Research Updates." CAR T-Cell Immunotherapy for ALL. National Cancer Institute, n.d. Web. 15 Apr. 2014. <http://www.cancer.gov/cancertopics/research-updates/2013/CAR-T-Cells>.